Molecular characterization of genetically modified (GM) crops has traditionally been accomplished... more Molecular characterization of genetically modified (GM) crops has traditionally been accomplished through a combination of Southern blot, polymerase chain reaction (PCR), and Sanger sequencing analyses. This characterization generates critical information that is used in downstream safety assessment of GM crops and development of GM detection methods.Next generation sequencing (NGS) technologies, such as whole genome sequencing (WGS), have shown the potential to replace some or all of these techniques for molecular characterization of GM crops. This paper describes the characterization of two GM maize events using NGS for WGS in combination with an insertion site characterization (ISC) method. The sensitivity of the method is also compared to that of Southern blot analysis through detection of small insert fragments. Our results demonstrate that WGS is at least as sensitive as Southern blot analysis for determining the insert copy number, presence or absence unintended insertions, ...
Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercia... more Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, t...
The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American f... more The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the
Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf... more Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families a...
Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf... more Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied across the scales of a function, to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. This approach does not only predict plant family, but also the collection site, confirming phylogenetically i...
Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facil... more Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also sh...
Cold-induced sweetening (CIS) is the accumulation of reducing sugars in potato tubers at low stor... more Cold-induced sweetening (CIS) is the accumulation of reducing sugars in potato tubers at low storage temperatures. It is undesirable because it results in dark fry products. Our study evaluated the relationship between genetic resistance to CIS and two starch parameters, amylose content and starch granule size. We found that the amylose content in four CIS-resistant varieties was higher than that in five susceptible varieties. Amylose content was influenced not only by variety but also storage, production year, and field location. However, interactions between amylose content and environmental variables were not detected. In contrast, starch granule size was not associated with CIS resistance. No effect of storage on starch granule size was detected, and interactions among variety, production year, and field location were observed. Tuber starch amylose content should be considered a source of variability for CIS.
This protocol describes a high through put colorimetric method that relies on the formation of a ... more This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.
The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American f... more The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the
The complete plastid genome sequence of the American cranberry (Vaccinium macrocarpon Ait.) was r... more The complete plastid genome sequence of the American cranberry (Vaccinium macrocarpon Ait.) was reconstructed using next-generation sequencing data by in silico procedures. We used Roche 454 shotgun sequence data to isolate cranberry plastid-specific sequences of "HyRed" via homology comparisons with complete sequences from several species available at the National Center for Biotechnology Information database. Eleven cranberry plastid contigs were selected for the construction of the plastid genome-based homologies and on raw reads flowing through contigs and connection information. We assembled and annotated a cranberry plastid genome (82,284 reads; 185x coverage) with a length of 176 kb and the typical structure found in plants, but with several structural rearrangements in the large single-copy region when compared to other plastid asterid genomes. To evaluate the reliability of the sequence data, phylogenetic analysis of 30 species outside the order Ericales (with 54 genes) showed Vaccinium inside the clade Asteridae, as reported in other studies using single genes. The cranberry plastid genome sequence will allow the accumulation of critical data useful for breeding and a suite of other genetic studies.
Background: The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-culti... more Background: The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America-the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear. Results: The genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%). Conclusions: Shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.
This is the first de novo assembly and annotation of a complete mitochondrial genome in the Erica... more This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.
In a number of ways, modern potato breeding efforts differ from those that created heirloom culti... more In a number of ways, modern potato breeding efforts differ from those that created heirloom cultivars. As a result of the rapid expansion of the potato processing industry in the mid twentieth century, potato breeders shifted their focus from fresh market varieties to those intended for the fry and chip markets. Selection for higher dry matter content and lower reducing sugar levels in tubers was successful. This study sought to determine whether modern processing cultivars differ from heirloom cultivars for two major characteristics of tuber starch, amylose content and starch granule morphology. Since breeding efforts for processing potatoes have focused on the maintenance of tuber quality during storage, these parameters were measured in both fresh and stored tubers. Twenty cultivars selected to span the range of cultivar release dates in the U.S. were grown at Hancock, WI in 2009 and 2010 and evaluated for tuber amylose content and measurements of starch granule surface area, length, width, length to width ratio, and smoothness. These traits were found to be stable across years and during storage. Significant differences in the amylose content and starch granule morphology exist among cultivars, but there is no clear distinction between modern and heirloom cultivars. Genetic variation for amylose content and starch granule morphology was found
Sugarbeet is a major source of refined sucrose and increasingly grown for biofuel production. Dem... more Sugarbeet is a major source of refined sucrose and increasingly grown for biofuel production. Demand for higher productivity for this crop requires greater knowledge of sugarbeet physiology, pathology, and genetics, which can be advanced by the development of new genomic resources. Towards this end, a sugarbeet transcriptome of expressed genes from leaf and root tissues at varying stages of development and production, and after elicitation with jasmonic acid (JA) or salicylic acid (SA), was constructed and used to generate simple sequence repeat (SSR) markers. The transcriptome was generated via paired‐end RNA sequencing and contains 82,404 unigenes. A total of 37,207 unigenes were annotated, of which 9480 were functionally classified using clusters of orthologous groups (COG) annotations, 17,191 were classified into biological process, molecular function, or cellular component using gene ontology (GO) terms, and 17,409 were assigned to 126 metabolic pathways using Kyoto Encyclopedi...
Caracterización molecular para el manejo de la colección colombiana de musaceas
The Musa germplasm bank established in Colombia has accessions tolerant to Yellow Sigatoka and is... more The Musa germplasm bank established in Colombia has accessions tolerant to Yellow Sigatoka and is located at over 1310 meters above sea level. This germplasm has been characterized using morphological descriptors. However it is has been difficult to identify some ...
Molecular characterization of genetically modified (GM) crops has traditionally been accomplished... more Molecular characterization of genetically modified (GM) crops has traditionally been accomplished through a combination of Southern blot, polymerase chain reaction (PCR), and Sanger sequencing analyses. This characterization generates critical information that is used in downstream safety assessment of GM crops and development of GM detection methods. Next generation sequencing (NGS) technologies, such as whole genome sequencing (WGS), have shown the potential to replace some or all of these techniques for molecular characterization of GM crops. This paper describes the characterization of two GM maize events using NGS for WGS in combination with an insertion site characterization (ISC) method. The sensitivity of the method is also compared to that of Southern blot analysis through detection of small insert fragments. Our results demonstrate that WGS is at least as sensitive as Southern blot analysis for determining the insert copy number, presence or absence unintended insertions, and for characterization of small fragment insertions. These results support the conclusion that WGS along with an appropriate insertion site characterization method are a suitable alternative to Southern blot analyses for molecular characterization of GM maize.
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Papers by Diego Fajardo